Effect of plant VOCs and light intensity on growth and reproduction performance of an invasive and a native Phytolacca species in China

Abstract Invasive plants often pose great threats to the growth of co‐occurring native plant species. Identifying environmental factors that facilitate exotic plant invasion and native species decline are important. In this study, we measured the effects of plant volatile organic compounds (VOCs), light intensity, and their interactions on the growth and reproduction performance of indigenous Phytolacca acinosa, and invasive Phytolacca americana, which has largely replaced the former in China. VOCs of invasive P. americana and low light levels both had negative effects on P. acinosa morphological and reproductive traits (stem length, average leaf number, total number, and length of racemes), and biomass allocation (total biomass, and leaf and flower mass fraction); low light also affected photosynthesis‐related trait (specific leaf area) of P. acinosa. In contrast, VOCs of P. acinosa had no significant effect on P. americana, but low light levels adversely affected its morphological and reproductive traits (stem length, total number, and length of racemes) and biomass allocation (total biomass, stem, and leaf mass fraction). Interactions between plant VOCs and light intensity had no significant effects on P. acinosa or P. americana. Under all experimental treatments, stem length, average leaf area, total number, and length of racemes, Root/Shoot ratio, root and flower mass fraction of P. americana were higher than those of P. acinosa, while average leaf number, specific leaf area, and leaf mass fraction was lower. These results indicated that P. acinosa was sensitive to P. americana VOCs and low light, which might affect the growth of sympatric P. acinosa. P. americana was negatively influenced by low light, but higher plant height and more reproductive organ resource allocation relative to sympatric P. acinosa might contribute to invasion success.


| INTRODUC TI ON
Invasive plants often exhibit rapid growth and efficient reproduction (Assad et al., 2021;Cai et al., 2021;Luo et al., 2020;Skálová & Pyšek, 2009), and generally become dominant in the introduced range, causing serious ecological damage and economic loss (Bartz & Kowarik, 2019;Rai & Singh, 2020). Identifying environmental factors that facilitate alien plants invasion are important and helpful to understand invasion mechanisms further, and provide insights into local species protection.
The "novel weapons hypothesis" emphasizes that allelopathic compounds, for example, airborne plant volatile organic compounds (VOCs), secreted by alien plants contribute to invasion success (Barney et al., 2009;Callaway & Aschehoug, 2000;McBride et al., 2020). Plant VOCs are cues responsible for the communication among plants (Kegge & Pierik, 2010;Kigathi et al., 2019;Meents et al., 2019). The role plant VOCs play in the growth of sympatric plant species can be negative, neutral, or positive. VOCs emitted from aerial parts of invasive Xanthium italicum had a negative effect on the growth of surrounding plants, such as Amaranthus mangostanus, Lactuca sativa, and Triticum aestivum (Shao et al., 2013(Shao et al., , 2019, while root VOCs released by Centaurea stoebe had a neutral to positive influence on the growth of neighbor plants (Gfeller et al., 2018).
Clarifying the functions of species-specific VOCs on plant-plant interactions is essential in understanding of the influence of invasive plants on native ones.
Compared to native plant species, invasive plants are often better competitors for light and exhibit greater adaptability to light environments. Mikania micrantha had been shown to grow slowly under low light, but transition to rapid growth under high light, blocking the sunlight of competing vegetation (Liu, Yan, et al., 2020;Liu, Chen, et al., 2020;Shen et al., 2015). Invasive Rhododendron ponticum could form larger leaf area to improve photosynthetic potential and lightharvesting efficiency than non-invasive Ilex aquifolium (Niinemets et al., 2003). Lantana camara adapted to moderately shaded environments by increasing leaf size, leaf biomass, and plant height (Carrión-Tacuri et al., 2011). Moreover, light is associated with the production of plant VOCs (Gouinguené & Turlings, 2002). Seasonal change or photoperiod, which is closely related to light intensity, could affect plant volatiles emission (Effah, Barrett, Peterson, Godfrey, et al., 2020;Hansen & Seufert, 2003;Tarvainen et al., 2005). Therefore, measurements of the interactions between plant VOCs and light intensity on invasion success are needed.
Phytolacca americana L., an herbaceous perennial shrub, was introduced from North America in 1935 and invaded most areas of central and southern China (Xu et al., 2012). Poisoning accidents would take place if P. americana was taken in inappropriately (Kim et al., 2005). Indigenous Phytolacca acinosa Roxb., a traditional Chinese medicinal plant, has been largely replaced by invasive P. americana (Xiao et al., 2019). Co-occuring P. americana and P. acinosa showed great difference in the natural environment ( Figure S1), leading to questions as to the factors that led to the decrease of P. acinosa and invasion success of P. americana. Here, plant VOCs released by aerial parts and roots of P. acinosa and P. americana, together with light intensity of 3000 lux, 2000 lux, and 1500 lux were employed to test their influence on morphological, physiological, and reproductive traits as well as resource allocation of P. acinosa and P. americana.
These results would illustrate the influence of plant VOCs and light intensity on the growth and reproductive performance of native P. acinosa and invasive P. americana, and provide insights into abiotic and biotic invasion mechanisms.

| Plant materials
Mature racemes in the canopy of P. americana and P. acinosa were collected randomly from 15 plants in Kunming (24º49ʹ N,102º52ʹ E) and Qujing (25º26ʹ N, 104º19ʹ E), Yunnan, China, respectively, in August, 2020. Seeds were achieved after removing the fleshly tissue of the fruits. For germinating, seeds were treated with H 2 SO 4 to break dormancy first, and then were cultured on 1% agar medium in an artificial incubator (Yiheng, Shanghai, China). Growth conditions were set at 27ºC for 14 h light and 25ºC for 10 h dark, approximating the natural growing season of Kunming, Yunnan province (generally in April-July) except the temperature was approximate to the average temperature in the day (Liu, Yan, et al., 2020;Liu, Chen, et al., 2020). To avoid the interference of VOCs, germinating seeds of each species were cultured separately in sealed plastic boxes (13 cm × 8 cm × 6.5 cm) on 100 ml of 1% agar medium. Seeds germinated within 24 h were transferred into another set of sealed plastic boxes with soil (Jiangsu Peilei Matrix Technology Development Co., Ltd., China) for seedling establishment. After the third leaf emerged, seedlings were transplanted into pots (bottom diameter = 9 cm, top diameter = 12 cm, height = 12 cm) filled with the same soil as described above for one seedling per pot, and subjected to different treatments immediately.

| Experimental design
We created a completely randomized design with the following factors: plant VOCs (VOCs of P. acinosa or P. americana, or no extra VOCs), light intensity (3000 lux, 2000 lux, or 1500 lux).
To investigate the influence of plant VOCs on the performance of neighboring plants, VOCs produced by five plants of P. acinosa or P. americana were used separately, and no extra VOCs was set as the control. To estimate the effect of light intensity on plant performance, light intensity of 3000 lux, which is similar with the natural light environment of P. acinosa accepted in the shade of P. americana at 10 AM in the growth season, was employed as a control, and 2000 lux and 1500 lux were tested individually to represent low light environments. Plants of P. acinosa and P. americana were randomly exposed to the tested VOCs, light intensity, or their interactions, respectively. Specifically, five plants of P.
acinosa grown under 3000 lux were treated as control, with each plant separated by about 8 cm. For plant VOC treatments, five seedlings of P. acinosa were co-cultured with another five P. acinosa under 3000 lux, and another five plants of P. acinosa were also alternatively placed with five P. americana under 3000 lux. Simultaneously, same treatments described above were also conducted on P. americana. A diagram describing the experimental design was presented in Figure S2. Each treatment with five plants was repeated three times. Sufficient water was equally provided to each plant, and other environmental factors were kept the same to ensure normal growth of the plants.
In order to test the influence of VOCs on plant growth effectively, the confined space provided by artificial chambers was used. Moreover, the light intensity of the artificial chamber was precisely maintained. The space of the chamber was divided equally into three sections with the light intensity of 1500 lux, 2000 lux, and 3000 lux, respectively, which was monitored by an illumination photometer (LBJ-20; Hangzhou Lvbo Instrument Co., Ltd, China).

| Plant trait measurements
Total chlorophyll content of the second fully expanded leaf was determined by an UV-330 spectrophotometer (0.3 × 0.3 cm 2 window area; Hangzhou Lvbo Instrument Co., Ltd, China). All leaves of each plant were scanned (CanoScan LiDE 300, Canon, Japan), and their respective leaf areas were measured using ImageJ (http://rsbweb.nih.gov/ij/). Average leaf area was calculated using the total leaf area and the total number of leaves per plant. The lengths of the stem and raceme were measured with a ruler. Plants were removed from the soil and cleaned before leaves, stems, racemes, and roots of each plant were dried to a constant weight (70°C for 72 h) as measured by an analytical balance (BSA223S, OLABO, Shandong, China) with an accuracy of 0.001 g. Specific leaf area (SLA) was calculated by the total leaf area and the total leaf biomass. Root biomass and shoot biomass (leaf biomass + stem biomass + raceme biomass) were used to obtain the root/shoot (R/S) ratio . Resource allocation was assessed by the biomass of a particular plant tissue and the total biomass of the plant according to the formula: leaf mass fraction (LMF) = leaf biomass/total biomass of the plant × 100% (Assad et al., 2021); stem, flower, and root mass fraction (SMF, FMF, and RMF) was similarly calculated.

| Data analysis
To test the influence of plant VOCs, light intensity, and their interactions on the morphological, physiological, and reproductive traits, together with biomass allocation of P. acinosa and P. americana, we used two-way ANOVA (implemented in SPSS 16.0, Chicago, IL, USA) based on General Linear Models (GLMs). Before analysis, normality and homogeneity of variances were tested, and log-transformation of the data was performed to correct deviation if necessary. Fisher's least significant difference (LSD) tests were employed to detect significant difference among treatments. Independent-sample t-test was also used to analyze differences between P. acinosa and P. americana.

| RE SULTS
According to the two-way ANOVA analysis, plant VOCs and light intensity had different effects on plant traits of P. acinosa and P. americana, but their interactions did not (Table 1). Morphological traits, including stem length and average leaf number, reproductive traits, including total number and length of racemes, together with biomass allocation which including total biomass, LMF, and FMF of P. acinosa were affected not only by low light level but also by the VOCs of P.
americana. Low light level also influenced SLA, R/S ratio, and RMF of P. acinosa, and the VOCs of P. americana also impacted its SMF. Similarly, the stem length, total number of racemes, the length of the 1st and 2nd racemes, total biomass, SMF, and LMF of P. americana were affected by low light level, but the VOCs of P. acinosa had no significant effect on P. americana. The relative content of chlorophyll, however, was not affected by the VOCs of neighbor plants or low light level.
For plant morphological traits, stem length of P. americana was statistically longer than that of P. acinosa (Table S1). Low light resulted in short stems in both plant species, and the stem length of P. acinosa grown under low light was negatively affected by the VOCs of P. americana (Figure 1a). Average leaf number of P. acinosa was significantly higher than P. americana's (Table S1). The leaf number of P. acinosa decreased when light intensity was reduced, while the leaf number of P. americana was not significantly different under any treatment. Furthermore, leaf number of P. acinosa under low light level decreased when exposed to VOCs of P. americana (Figure 1b).
When taken photosynthesis-related traits into consideration, there were no significant differences in average leaf areas of either species under the treatments of plant VOCs, light intensity, or their interactions, but the average leaf area of P. acinosa was statistically smaller than that of P. americana (Figure 1c; Table S1). Low light level significantly increased the SLA of P. acinosa, whether the VOCs of P. acinosa or P. americana were provided or not. However, SLA of P.
americana was not significantly affected by plant VOCs or light intensity, and was smaller than P. acinosa (Figure 1d; Table S1). Relative content of chlorophyll of either species was not statistically different under any treatment (Figure 1e).
The reproductive traits were also showed difference among these treatments. No more than one raceme was produced by P. acinosa, while up to three were produced by P. americana. The total number of racemes of P. americana was statistically greater than P. acinosa, and low light level had a negative effect on raceme production of both species (Figure 2a; Table S1). The length of the first raceme of P. americana was significantly longer than P. acinosa, and racemes of both species shortened as light intensity decreased. The VOCs of P. americana also had a negative effect on the raceme length of P. acinosa (Figure 2b; Table S1). The second and the third raceme only found in P. americana, and the length of them both decreased as the light intensity reduced (Figure 2c-d; Table S1).
The total biomass of both species significantly decreased with the reduced light level. The total biomass of P. acinosa was also negatively affected by the VOCs of P. americana. Except for plants grown under 3000 lux, or the combination of 3000 lux and the VOCs of P. acinosa, P. acinosa generally had lower total biomass than P. americana (Figure 3a; Table S1). The R/S ratio of P. americana was significantly higher than P. acinosa, and the R/S ratio of P. americana was slightly increased as light intensity decreased, while it significantly decreased in P. acinosa (Figure 3b; Table S1).
Similarly, the RMF of P. americana was statistically higher than P. acinosa, and the RMF of P. americana increased as light intensity decreased, while it decreased greatly in P. acinosa (Figure 3c; Table   S1). SMF of P. americana under 1500 lux was lower than P. acinosa, while it was higher than P. acinosa under the combination of 3000 lux and the VOCs of P. americana. Under the treatment of TA B L E 1 Two-way ANOVA analysis of the effects of VOCs and light intensity on plant traits of Phytolacca acinosa and Phytolacca americana 1500 lux, the SMF of P. acinosa was decreased when exposed to the VOCs of P. americana. In P. americana, SMF decreased significantly under low light levels ( Figure 3d; Table S1). LMF of P. acinosa was significantly higher than that in P. americana, and low light intensity increased the resource allocation to leaves of P. acinosa and P. americana. Under the treatment of 1500 lux, LMF of P. acinosa increased when exposed to the VOCs of P. americana (Figure 3e; Table S1). FMF of P. americana was significantly higher than P. acinosa, and low light level decreased FMF of both P. acinosa and P. americana (Figure 3f; Table S1).

| DISCUSS ION
Plant morphological and reproductive traits, together with biomass allocation, of native P. acinosa were affected by both the VOCs of invasive P. americana and light intensity. Low light level also affected photosynthesis-related trait of P. acinosa, but for invasive P. americana, only low light level had effects on morphological and reproductive traits as well as biomass allocation. Generally, P. americana exhibited growth and reproduction advantages than P. acinosa, including longer stem, larger leaf area, greater numbers of racemes, larger total biomass, higher R/S ratio, and higher FMF, which, together, likely enhance its invasiveness, especially when in concert with its VOCs and at low light levels.

| Invasive P. americana grown better than native P. acinosa under the effect of plant VOCs and low light level
Taller plants often capture more resources, especially light, for growth (Thomson et al., 2011), and low light level had negative impacts on the growth of P. acinosa and P. americana. VOCs of P. americana negatively affected the growth of P. acinosa, resulting in shorter stems, reduced total biomass, and LMF. About 50% aboveground biomass of Solidago canadensis was reduced by VOCs of invasive Artemisia vulgaris (Barney et al., 2009). In general, invasive species are taller than co-occurring native species (Assad et al., 2021;Wang et al., 2018).
We found that the stem length of invasive P. americana was also longer than native P. acinosa under all treatments, but the leaf number of P. americana was lower. Shorter stem length and higher leaf number suggested that native P. acinosa had higher leaf density than invasive P. americana, which might indicate that P. acinosa invested more resources in leaf construction (Feng et al., 2007). Similar results were also found that P. acinosa had higher LMF than P. americana.
High SLA is often positively correlated with high ability of resource capture, such as carbon assimilation and carbon allocation (te Beest et al., 2015;Wang et al., 2018). Invasive plants generally have larger SLA than non-invasive plants, but similar or lower SLA of invasive species were also found (Grotkopp et al., 2010;Qin et al., 2012;van Kleunen et al., 2010;Wang et al., 2017). Native P. acinosa was more sensitive to low light level as its SLA fluctuated significantly with the changed light intensity. Even though P. acinosa and P.  (Feng et al., 2007;He et al., 2018).
4.2 | Invasive P. americana showed a reproduction advantage than native P. acinosa when exposed to plant VOCs and low light intensities Invasive plants tend to exhibit higher reproductive capacity than natives in order to become dominant in the community and invade  Figure 1 habitats (Assad et al., 2021). High light level could increase the seed production of invaders (Feng et al., 2007). Invasive P. americana had more racemes than native P. acinosa, and high light level treatment increased the production of racemes of both species. The first raceme of P. americana was significantly longer than that of P. acinosa, and many P. americana produced three racemes, while P. acinosa produced no more than one. Accordingly, FMF of P. americana was higher than P. acinosa, consistent with a previous study of invasive Rubus discolor, which allocated more resources to the reproductive organs than non-invasive Rubus ursinus (McDowell & Turner, 2002).
Moreover, roots were essential for perennial plants that biomass allocation to roots made perennial weeds difficult to control (Ringselle et al., 2017). Even though invasive P. americana and native P. acinosa had similar total biomass, P. americana had higher R/S ratio and RMF than P. acinosa under all treatments. The biomass allocation to belowground or aboveground part is a trade-off of plant to uptake water and nutrient from the soil or capture light resource, and invasive species are characterized by high resource acquisition (Feng et al., 2007;Funk, 2013;Wang et al., 2018). The biomass allocation patterns of P. americana may further enhance its invasiveness.

| CON CLUS ION
VOCs of the invasive P. americana adversely affected the growth performance of the native P. acinosa, as measured by stem length, average leaf number, total biomass, and LMF. Low light levels had a negative effect on the performance of both P. americana and P. acinosa, resulting in shorter stem length, lower total number and length of racemes, lower total biomass, and resource allocation. In the natural environment, P. americana are generally taller than sympatric P.
acinosa, and VOCs emitted by P. americana and shade environment formed by P. americana may reduce P. acinosa growth. However, the particular volatile released by P. americana that influence the performance of P. acinosa needs to study further.

ACK N OWLED G M ENTS
The authors thank Dr. Ian Gilman at Yale University for his assistance with English language and grammatical editing. They thank Miss.

CO N FLI C T O F I NTE R E S T
The authors declare that there have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data of the study is available at https://doi.org/10.5061/ dryad.bnzs7 h4cf.